Genomic, morphological, and biochemical analyses of a multi-metal resistant but multi-drug susceptible strain of Bordetella petrii from hospital soil

Contamination of soil by antibiotics and heavy metals originating from hospital facilities has emerged as a major cause for the development of resistant microbes. We collected soil samples surrounding a hospital effluent and measured the resistance of bacterial isolates against multiple antibiotics and heavy metals. One strain BMCSI 3 was found to be sensitive to all tested antibiotics. However, it was resistant to many heavy metals and metalloids like cadmium, chromium, copper, mercury, arsenic, and others. This strain was motile and potentially spore-forming. Whole-genome shotgun assembly of BMCSI 3 produced 4.95 Mb genome with 4,638 protein-coding genes. The taxonomic and phylogenetic analysis revealed it, to be a Bordetella petrii strain. Multiple genomic islands carrying mobile genetic elements; coding for heavy metal resistant genes, response regulators or transcription factors, transporters, and multi-drug efflux pumps were identified from the genome. A comparative genomic analysis of BMCSI 3 with annotated genomes of other free-living B. petrii revealed the presence of multiple transposable elements and several genes involved in stress response and metabolism. This study provides insights into how genomic reorganization and plasticity results in evolution of heavy metals resistance by acquiring genes from its natural environment.


Results and discussion
Isolation, multi-drug and multi-metal tolerance of strain BMCSI 3 from soil. Four individual bacterial colonies grew on Luria-Bertani Agar plate of which one was designated as BMCSI 3. First, we tested its sensitivity against multiple antibiotics. It was found to be sensitive to all the tested antibiotics including Amino benzyl penicillin, Penicillin, Penicillin beta-lactam, 3 rd and 4 th generation Cephalosporins, Amino Glycosides, Tetracyclines, Fluoroquinolone, Macrolides, Quinolone, and Sulphonamide group of antibiotics (Fig. 1a). Using X-ray fluorescence spectroscopy (XRF), we determined that the soil was contaminated with multiple toxic metals like Titanium (Ti), Iron (Fe), Copper (Cu), Zinc (Zn), Molybdenum (Mo), Cadmium (Cd), and Lead (Pb). The presence of Potassium (K) and Calcium (Ca) were also detected from XRF spectroscopy. Therefore, we tested the growth of BMCSI 3 in the presence of toxic heavy metals. BMCSI 3 grew in the presence of very high concentrations of salts of Lead, Molybdenum, Manganese (⁓3000 mg/L), Copper (⁓1000 mg/L), and Iron (⁓750 mg/L) (Fig. 1b). In comparison to these heavy metals, BMCSI 3 tolerated lower concentrations (⁓10 mg/L) of Chromium, Cobalt, Arsenic (III and V) salts and did not grow in the presence of Zinc, Cadmium and Mercury (Fig. 1c). However, by analyzing these results, in one way, BMCSI 3 is susceptible to antibiotics, other way, it is highly resistant to multiple metals.
Whole-genome sequences and phylogenomic inference. The draft genome of BMCSI 3 was assembled using the three genome assemblers, SPAdes, Velvet, and ABySS; the latter two were based on the best suited K-mer statistics. For instance, the best assembly statistics were obtained with K-mer length of 99 in Velvet and k-mer length of 96 in ABySS. The contigs produced by the SPAdes, Velvet, and ABySS programs are 66, 14, and 114, respectively. The comparative assembly statistics using the QUAST software revealed that the assemblies consisted of approximately 4.9 Mb genome with the highest N50 length of 3.8 Mb was found with Velvet assembly, which was finally processed for further analysis. The finalized draft assemblies of BMCSI 3 were 99.53% complete without any contaminations (Supp. file S1).
Whole-genome sequence of BMCSI 3 was analyzed to predict its taxonomic status through different web tools. As per the predictions made by MiGA, StrainSeeker, and TYGS webservers, the closest relative determined was B. petrii DSM 12804 T . The tree inferred with FastME 2.1.6.1 19 from GBDP (Genome BLAST Distance Phylogeny) distances calculated from genome sequences. The branch lengths were scaled in terms of GBDP distance formula d 5 . The numbers above branches are GBDP pseudo-bootstrap support values > 60% from 100 replications, with average branch support of 97.9%. The tree was rooted at the midpoint 20 (Fig. 2a). The taxonomic classification was thus resolved to B. petrii under the family Alcaligenaceae, order Burkholderiales, and phylum Proteobacteria. Different features of B. petrii BMCSI 3 genome were analyzed including the coding sequence of genes and GC content distributed across the scaffolds. From outermost to centre, distribution of scaffolds (Ring-1); protein-coding genes (CDS) in forward and reverse strand (Ring-2 and 3); Blastp hit with a reference B. petrii DSM 12804 T genome (Ring-4); GC skew plot with a value on above average and below average (Ring-5); and plots of GC content (Ring-6) (Fig. 2b).
Genome features. BMCSI 3 contains a total of 4,708 genes of which 4,638 were protein-coding genes (CDS) and 41 were pseudogenes. The number of genes that encode tRNAs, rRNAs, and ncRNAs were found to be 54, 12, and 4, respectively. Annotation by RAST resulted in 29% (1337) of the protein-coding genes being covered under the 324 subsystems. The metabolism of amino acids and derivatives occupied the largest number     21 was identified in the genome of metal-loving BMCSI 3. Presence of proteins like heavy metal sensor, response regulator and efflux system could also influence heavy metal resistant 22,23 . The presence of two-component response regulators and multiple genes with potential function in heavy metal resistance resulted in successful adaptation and survival of this strain in an environment that is heavily contaminated with toxic metals 24 .
Morphology and biochemical features. BMCSI 3 is a Gram-negative, rod-shaped bacterium. This strain tested positive for catalase, amylase, lipase and utilized citrate as carbon source. A total of 14 proteincoding genes related to flagella under the category of motility and chemotaxis were identified including flagellar biosynthesis proteins like FlhA, FlhB, FliR, FlhF; motor switch protein FliM, FliN; motor rotation protein MotA, MotB; basal-body rod modification protein FlgD; L-ring protein FlgH. Other chemotaxis related proteins, like CheR, CheW, CheB, CheA, response regulator CheY including a type IV pili methyl-accepting chemotaxis transducer N-terminal domain-containing protein that usually mediates chemotactic response were also found to be present 25 . Generally, Bordetella species sense environmental cues and control transcription of virulence genes by the activity of BvgAS (Bordetella virulence gene) system. When BvgAS is inactive, Bordetella produce functional flagella and thus are motile. The genome of BMCSI 3 lacks genes homologous to bvgA and bvgS 26 . Based on these findings, we hypothesized that BMCSI 3 will be flagellated. Consistent with this hypothesis, transmission electron micrographs revealed peritrichous flagellation and ⁓8-10 µm elongated mature vegetative cells (Fig. 3a-c). A forespore-like formation and cell outburst was observed within the mature vegetative cell of BMCSI 3 (Fig. 3c). Free endospore-like structures (Fig. 3d) were also observed. To date, the phenotype of sporulation has www.nature.com/scientificreports/ not been reported in Bordetella. Additionally, micrographs clarifying the cell morphology are not available for B. petrii 27 . In the draft genome of BMCSI 3, spoVC that encodes peptidyl-tRNA hydrolase and is essential for vegetative growth was present along with spore maturation proteins 28 . However, genes homologous to sporulationspecific sigma factors of Bacillus species were absent 29-31 .  abundant IS3 family including ISL3, IS5,  IS21, IS66, IS91, IS110, IS360, IS1066, and IS1663 families (Fig. 4). Consistent with previously published report, the repetitive element IS481, which is a frequent target of diagnosis of other Bordetella species was not found in BMCSI 3 27,32 . Most of the putative laterally acquired GIs found in BMCSI 3 harbored heavy metal resistant genes, multiple response regulators and other transcription factors, transporters, and multi-drug efflux pumps (Supp. file S2). GI-1 contains a TniQ family protein having a role in transposition of the mercury-resistance transposon Tn5053 33 . GI-1 and 2 harbours genes having homology to the ZorAB system, which is involved in the opening of the proton pump leading to the membrane depolarization upon phage infection and ensuring abortive transduction 34 . This may lower the chances of acquiring new genes, especially antibiotic resistance genes into the bacterial genome. Consistent with this, BMCSI 3, has genes that are usually associated with antibiotic resistance, such as small multidrug resistance (SMR) and resistance-nodulation-division (RND) antibiotic efflux pump 35 .

Genomic islands.
Interestingly, no such cross-resistance link between heavy metals resistance and antibiotic resistance was found. Eventually, almost all the SMR and RND groups of efflux pump systems are likely to involve metals resistance, instead of antibiotic resistance during different environmental stresses (Fig. 1). HlyD, an important component of CusCFA efflux membrane fusion protein is present in multiple GIs. This type of resistance-nodulation-division group (RND) has evolved to secrete toxic metallic ions outside the periplasmic space 36 . MFS transporters involved in both efflux and influx of various substances including drug efflux from the cell are also present in the genome 37 . Another major uncharacterized transporter belonging to the DMT family of transporters is also integrated into the genome. GI-6 and GI-42 harboured arsenical resistance protein-coding gene arsH, Cd(II)/ Pb(II)-responsive transcriptional regulator protein-coding gene cadR, arsenate reductase protein-coding gene arsC, and ACR3 family arsenite efflux transporter related gene arsB. GI-44 harbors multicopper oxidase domaincontaining protein, copper-translocating P-type ATPase, efflux RND transporter periplasmic adaptor and permease subunit, copper-binding protein, metal-binding protein, copper homeostasis membrane and periplasmic binding protein, copper resistance protein B, copper resistance system multicopper oxidase, heavy metal sensor histidine kinase, heavy metal response regulator transcription factor, CusA/CzcA family heavy metal efflux RND www.nature.com/scientificreports/ transporter, and periplasmic adaptor subunit. GI-33 acquired a universal stress protein that may regulate a broad range of cellular responses against biotic and abiotic stress 38 . Additionally, GI-44 harboured the gene encoding for the copper-translocating P-type ATPase whose primary function is to transport copper across the biological membrane 39 . Copper homeostasis membrane protein CopD, periplasmic protein CopC, copper resistant protein B, multicopper oxidase are also present in the same genomic island which may promote tolerance in the presence of copper. A MATE family efflux transporter CusA/CzcA family heavy metal efflux is present in GI-44. Additionally, genes encoding heavy metal sensor histidine kinase and heavy metal response regulator transcription factor are integrated within the GI-44 at multiple sites.
Genome comparison. Bacteria belonging to the genus Bordetella are gram-negative coccobacilli of the phylum proteobacteria. Most species in this genus are capable of causing a wide spectrum of pulmonary and bronchial diseases, in humans, animals, and birds 40,41 . To date, of the described nine species, B. pertussis and B. parapertussis cause whooping cough in humans 42 , while B. bronchiseptica infects primarily animals, and rarely humans 43 . Despite the fact that, evolutionary trend analysis demonstrated that the ancestors of this genus were of environmental origin, a significant loss of metabolic genes and acquisition or retention of virulence factors within the genome has driven this particular genus to emerge as an opportunistic pathogen 44,45 (Fig. 5). All the sixteen genomes of different species of Bordetella were compared along with the available strains of B. petrii using B. petrii DSM 12804 as a reference. From outermost to centre (Fig. 6) 47 , arcD (arginine/ornithine antiporter), areA (Nitrogen regulatory proteins which are GATA type transcription factors) 48 , hdfR (encodes a LysR family protein) 49 , cysIJ, hemN (oxygen-independent coproporphyrinogen-III oxidases) 50 , nrdD (Anaerobic ribonucleoside-triphosphate reductase activating protein) 51 , yhbU, ptlB, nimT (2-nitroimidazole transporter and similar proteins of the Major Facilitator Superfamily of transporters) 52 , tmoT, NADH dehydrogenase like protein coding gene, sasA (Adaptive-response sensory-kinase) 53 , yheG putative multidrug efflux transporter coding gene, norB, qoxC (subunit III of the aa3-type quinone oxidase) 54 , nirBQST, mftC, eysG, ccmH (Cytochrome C biogenesis protein) 55 , zupT (Zinc transporter) 56 , cusAB, dsbD, cnrA (membrane-bound protein complex catalyzing an energy-dependent efflux of Ni 2+ and Co 2+ ) 57 , hcaR, gcd, iolG, wbpl, mshA, pglK (six-transmembrane helical domain of the ABC transporter) 58 were only found within the genome of B. petrii. Interestingly, virulence regulon transcriptional activator related gene virB was only found within the genomes of B. petrii (Supp. file S4). Next, we carried out a comparative genome analysis of B. petrii. To date, six soil-born strains of B. petrii whose genome has been sequenced ( Table 1) of which only the Type Strain B. petrii DSM 12804 with a complete genome was reported from a river sediment enriched dechlorinating bioreactor 27 . Other strains viz., B. petrii J49 and B. petrii J51 were reported from aquatic soil and strain BT 1 9.2 was reported from contaminated soil. Another strain MY 10 was also reported from soil 59 . Genome sizes of these other strains ranged from 4.21 to 6.10 Mb, with an average GC% ranging from 65.4 to 67.3. The 4.95 Mb genome of BMCSI 3 was smaller than three other B. petrii strains and the overall GC content of 67.3% was the second highest among the six sequenced strains. The different categories of the genes present in the five genomes are shown in Table 1. Both BMCSI 3 and BT 1 9.2 isolated from contaminated soil harboured a significantly large number of heavy metal response and efflux transporter related proteins, but a smaller number of ABC transporter, virulence, motility and chemotaxis related proteins compare to the other four strains. Only the strain BT 1 9.2 is devoid of any type IV pili methyl-accepting chemotaxis transducer protein related to chemotaxis. Transmembrane signaling receptor PAS domain-containing methyl-accepting chemotaxis protein was found within strain DSM 12804, J49 and BMCSI 3. Two SOS response-associated peptidase family proteins were found within the chromosome of strain DSM 12804 but as a sign of rapid genome adaptation, this protein was acquired within the island of strain BMCSI 3 genome. Only three strains (J49, J51, and BT 1 9.2) of B. petrii harbour genes involved in sulfur metabolism and only two strains BMCSI 3 and DSM 12804 harbored the Type IV secretion system, multi-subunit secretion apparatus involved in secretion of macromolecules across the membranes. The highest numbers of tRNA were found within BMCSI 3 followed by DSM 12804. The decoding of mRNA into protein is governed by tRNA. Since www.nature.com/scientificreports/ the structural diversity of tRNA most likely co-evolved with their processing RNA splicing endonuclease 60 , it may be significant in the evolution of BMCSI 3. Pangenome analysis of six genomes of B. petrii represents 52,87,950 bp to be pangenome size including 10,69,661 bp core genes, 12,34,707 bp accessory genes and 29,83,582 bp strain-specific genes shared by at least 2 strains. So, the genome diversity of B. petrii represents an open pangenome model. However, functional analysis of strain-specific accessory and core genome suggests a usual core metabolism by those strains (Fig. 7).

Conclusion
Bacteria belonging to the genus Bordetella can cause a wide range of pulmonary and bronchial infections in humans, animals, and birds. However, B. petrii, a member of this genus has a wide spectrum of occurrence within the natural habitat. In this study, we report that a soil-inhabiting B. petrii strain isolated from soil sample adjacent to a hospital's effluent can tolerate toxic metalloids by acquiring different metal tolerant genes. Surprisingly, despite being of hospital origin, this strain was susceptible to multiple antibiotics. BMCSI 3 featured putative multidrug efflux transporters like MATE, MSF, RND, DMT, as well as ABC family proteins that are possibly involved in the effluxing of excess metal ions but not the drug molecules. The genomic islands represented a significant number of integrative and conjugative transposable elements. Moreover, genomic islands carried genes that encode metal efflux transporter (ACR3), copper resistant operon (Cop), and mercury-resistance transporter (mer) proteins that may result in metal resistance. Collectively, our results suggest that resistance to antibiotics and tolerance to metals are not always linked. A detailed phenotypic investigation of B. petrii is needed to understand the mechanisms through which microbes become resistant to heavy metals.  www.nature.com/scientificreports/ prepared broth and were allowed to incubate overnight at 30 °C, 130 rpm. Cell density was measured by the absorbance value taken at 600 nm using a Spectrophotometer (Lasany LI-721). Based on the multi-drug and multi-metal tolerance capability, strain BMCSI 3 was selected for this study.
Morphology and physicochemical study. Cell morphology of BMCSI 3 was studied by performing Transmission electron microscopy. Cell culture was drop cast onto a carbon-coated Cu grid (Sigma Aldrich), air-dried, and images were acquired under JEM 1400 plus, JEOL Transmission Electron Microscope (120 keV). Different physicochemical properties of strain BMCSI 3 like gram characteristics, extracellular enzyme activities (catalase, amylase, and protease), substrate hydrolysis (tributarin, gelatin, starch), and substrate utilization (lysine, citrate) were also performed 65 .
Genome sequencing. Cells of BMCSI 3 were cultured on Luria Bertani Agar media (HiMedia) at 30 °C, overnight and genomic DNA was extracted using the Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, USA). Final DNA concentration and purity were obtained using NanoDrop 1000 (Thermo Scientific, USA) and the genomic DNA integrity was checked on 1.5% agarose gel electrophoresis 66 . Approximately, 1 µg of genomic DNA was used to construct sequencing libraries by using the TruSeq™ DNA PCR-Free library preparation kit (Ilumina, Inc., USA). Before library preparation, the genomic DNA was fragmented using Covaris followed by end repair and adapter ligation. Finally, the sequencing was performed on Illumina MiSeq (Illumina, USA) platform.
Assembly and annotation. The raw paired-end fastq reads (2 × 301 bp) were quality checked using FastQC v.0.11.5 67 followed by trimming of low-quality bases in a sliding window approach using Trimmomatic v.0.39 68 . The cleaned reads were assembled separately using the SPAdes v.3.13.0 69 , VelvetOptimiser v.2.2.4 70 , and ABySS v.1.9.0 71 . The comparative evaluation of the assemblies generated from the three assemblers was carried out using the Quality Assessment Tool (QUAST v.5.0.2) 72 . The best assembly (with the highest N50 length) found with the QUAST analysis proceeded further for genome assembly-assisted bacterial strain identification using the MiGA webserver 73 , StrainSeeker 74 , and TYGS server 75 . The contigs from the Velvet assembly were processed for scaffolding and gap filling using the programs SSPACE v3.0 76 and GapFiller v.1.10 77 . Finally, the contigs were ordered using the Progressive Mauve 78 and a similar reference genome of Bordetella petrii DSM 12804 (NC_010170.1) to obtain the draft assembly of BMCSI 3. The assembly quality and genomic contaminations, if any, were evaluated using the CheckM-v.1.0.18 79 in KBase 80 . Further genomic analysis, annotation, and other comparative genomics studies were carried out using this BMCSI 3 draft assembly.
The contig assembly of BMCSI 3 was annotated using the software Prokka v.1.13.7 81 , Rapid Annotation System Technology (RAST) Pipeline 82 , PATRIC Pipeline 83 and National Center for Biotechnology Information (NCBI) stand-alone Prokaryotic Genome Annotation Pipeline 84 . The visualization of the genome and its typical features was carried out using the CGviewer server beta 85 . Genomic Islands within the genome of BMCSI 3 were predicted using IslandViewer 4 server 86 .